Non-targeted metabolomics analysis of exosomes derived from <italic>Glycyrrhiza uralensis</italic>

Exosomes are nanometer-sized secretory vesicles that play a pivotal role in mediating intercellular communication. Recently， exosomes derived from herbal medicine have garnered significant attention owing to their unique composition features and therapeutic potential. <italic>Glycyrrhiza uralensis</italic>， a traditional Chinese medicinal and edible plant， is rich in flavonoids， terpenoids， polyketides， and phenolic acids. However， the long-term consequences of overharvesting and artificial cultivation have led to inconsistent quality of licorice materials. Plant tissue culture technology offers a sustainable approach to preserve medicinal plant resources， enabling year-round production independent of seasonal and environmental constraints. To address these challenges and explore the therapeutic potential of herb-derived exosomes， this study employed a laboratory-constructed temporary immersion bioreactor system （TIBS） for <italic>G. uralensis</italic> cultivation and exosomes isolation. The key experimental procedures are summarized as follows： sterile buds derived from <italic>G. uralensis</italic> seeds were used as explants. The callus proliferation medium consisted of Murashige and Skoog medium （MSM） supplemented with 1.18 mg/L 6-benzylaminopurine （6-BA）， 1.44 mg/L naphthaleneacetic acid （NAA） and 1.44 mg/L 2，4-dichlorophenoxyacetic acid （2，4-D）， yielding a maximum proliferation coefficient of 10.81 after 21 days. For shoot proliferation， sterile plantlets were generated on MSM medium supplemented with 1 mg/L 6-BA， 0.05 mg/L NAA， and 150 mL/L coconut milk （CM）， which produced sterile plantlets within 28 days of culture. When cultured in the TIBS system with an immersion frequency of 5 min every 6 h， the callus proliferation coefficient reached 13.12 after 28 days of culture. Exosomes were successfully isolated from the TIBS-cultured <italic>G. uralensis</italic> calli using differential centrifugation and ultracentrifugation protocols. Comprehensive characterization was performed to validate the identity， purity， and stability of isolated exosomes. Morphological observation via transmission electron microscopy （TEM） revealed typical cup-shaped vesicles with a distinct lipid bilayer structure， which is consistent with the canonical exosomes morphology. Dynamic light scattering （DLS） analysis determined an average hydrodynamic diameter of 56 nm. The exosomes preparation exhibited a particle concentration of 1.07×10¹² particles/mL， and a zeta potential of （-15.030±3.815） mV， indicating excellent colloidal stability suitable for biological applications. To decipher the molecular cargo of these exosomes， untargeted metabolomic profiling was conducted. A total of 1 760 metabolites were identified across callus and exosomes samples， of which 1 068 differential metabolites （DMs） distinguished exosomes from parental calli. Notably， 195 DMs were significantly enriched in exosomes. KEGG （Kyoto Encyclopedia of Genes and Genomes） pathway enrichment analysis revealed enrichment in 11 metabolic pathways， primarily involving terpenoids， flavonoids， alkaloids， fatty acids， and phenylpropanoids. Bioactive compounds of interest included azelaic acid， phloretin， and senkyunolide C， highlighting their potential pharmacological significance. In summary， this study demonstrates that the TIBS-based in vitro platform enables efficient， scalable， and consistent production of <italic>G. uralensis</italic>-derived exosomes with homogeneous morphology and specialized metabolite profiles. The successful isolation， comprehensive characterization， and identification of bioactive metabolite enrichment establish a robust foundation for mechanistic investigations and translational applications. These exosomes exhibited promising therapeutic potential in anti-inflammatory， antioxidant， antitumor， and antibacterial biomedicine. Furthermore， this bioreactor system addresses the quality inconsistency issue in natural <italic>G. uralensis</italic> resources， paving new ways for developing innovative herbal exosome-based nanomedicines.